RPA alleviates the inhibitory effect of vinylphosphonate internucleotide linkages on DNA unwinding by BLM and WRN helicases
RPA alleviates the inhibitory effect of vinylphosphonate internucleotide linkages on DNA unwinding by BLM and WRN helicases
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DOI：
10.1093/nar/gkh709
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发表时间：
2004-07-01
影响因子：
14.9
通讯作者：
Janscak, P
中科院分区：
文献类型：
--
作者：
Garcia, PL;Bradley, G;Janscak, P
Bloom(BLM)和Werner(WRN)综合征蛋白是SF2 DNA解旋酶的RecQ家族成员。在本文中，我们证明了通过在转位的DNA链中引入乙烯基膦酸间的核苷酸间连接来限制DNA骨架的旋转灵活性，抑制了这些酶的有效的双链解离。人单链DNA结合蛋白复制蛋白A(RPA)完全恢复了BLM和WRN在含乙烯基膦酸底物上的解离活性，而来自大肠杆菌(SSB)的异源单链DNA结合蛋白仅部分恢复了该活性。RPA和SSB都不能恢复SF1 PcrA水解酶在修饰底物上的解离活性，这意味着RPA与BLM和WRN解旋酶具有特异性相互作用。我们的数据突出了SF1和SF2解旋酶之间的细微差异，并表明虽然RecQ解旋酶属于SF2家族，但它们在机制上更类似于SF1 PcrA解旋酶，而不是其他不受乙烯基膦酸酯修饰影响的SF2解旋酶。
Bloom (BLM) and Werner (WRN) syndrome proteins are members of the RecQ family of SF2 DNA helicases. In this paper, we show that restricting the rotational DNA backbone flexibility, by introducing vinyl phosphonate internucleotide linkages in the translocating DNA strand, inhibits efficient duplex unwinding by these enzymes. The human single-stranded DNA binding protein replication protein A (RPA) fully restores the unwinding activity of BLM and WRN on vinyl phosphonate-containing substrates while the heterologous single-stranded DNA binding protein from Escherichia coli (SSB) restores the activity only partially. Both RPA and SSB fail to restore the unwinding activity of the SF1 PcrA hellicase on modified substrates, implying specific interactions of RPA with the BLM and WRN helicases. Our data highlight subtle differences between SF1 and SF2 helicases and suggest that although RecQ helicases belong to the SF2 family, they are mechanistically more similar to the SF1 PcrA helicase than to other SF2 helicases that are not affected by vinylphosphonate modifications.